Gut Microbiome Associated with Cultured Malaysian Mahseer Tor Tambroides 1Shumpei Iehata, 1Mohamad Z

Gut microbiome associated with cultured Malaysian mahseer Tor tambroides 1Shumpei Iehata, 1Mohamad Z. M. Nosi, 2Muhd Danish-Daniel, 1Noor E. Sharifah 1 Faculty of Fisheries and Food Science, University of Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia; 2 Institute of Marine Biotechnology, University of Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia. Corresponding author: S. Iehata, [email protected] Abstract. In this study, 16S rRNA gene amplicon sequencing and PICRUSt analysis were carried out to better understand the cultured Malaysian mahseer (Tor tambroides) associated gut microbiome profile from three different culture farms. Principal Coordinates Analysis demonstrated that gut microbiome diversity had similar trend within same culture location and divergent among different location. 16S rRNA gene amplicon sequencing result showed that the dominant phylum such as Actinobacteria, Bacteroidetes, Firmicutes, and Fusobacteria was distinctly depending on their location, although phylum Proteobacteria was one of the core-gut bacterial community regardless of the location. Interestingly, some Propionibacteriaceae, which produce propionic acid by fermented plant materials, were detected only in Terengganu sample that fed only vegetables. Similarly, PICRUSt result showed the highest propionate metabolism in Terengganu. These results indicate that gut microbiome composition of T. tambroides seems to vary significantly among different culture locations and were highly dependent on their culture location, especially on their diet type. Furthermore, we found some beneficial bacterial DNA which have high possibility to be utilized as potential probiotics for T. tambroides culture. The present study provides new insights into the understanding on the gut microbiome structure of T. tambroides from different culture locations, and provides the relevant management information of combination of diet type and bacterial manipulation for development of sustainable aquaculture practices for this species, although we need further research on comparative analysis of the microbiome among gut and environmental sample. Key Words: 16S rRNA gene amplicon sequencing, linear discriminant analysis (LDA) effect size (LEfSe), Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), Propionibacteriaceae. Introduction. It is known that gut microbiota plays crucial role in host metabolism, nutrition, and health maintenance (Nicholson et al 2005; Nayak 2010). Moreover, Ramírez et al (2018) reported that understanding the gut bacterial community composition could give the relevant information on the fish diet requirements for the development of sustainable aquaculture. On the other hand, fish gut microbiota composition has been affected by several factors including diet composition, gut shape, host habitat and surrounding environment (McIntosh et al 2008; Sullam et al 2012; Wong & Rawls 2012; Eichmiller et al 2016; Egerton et al 2018). In particular, Ringø et al (2016) suggested that gut microbiota composition of aquatic animals are more sensitive to dietary changes than that of terrestrial animals. Similarly, Miyake et al (2015) indicated that diet type is strongly influenced on fish gut microbiota composition. Until now, it has been gradually revealed the fish bacterial community composition by means of NGS approach (Star et al 2013; Xia et al 2014; Ghanbari et al 2015; Li et al 2015; Li et al 2018b). In the recent years, the cyprinid gut microbiota composition and their diversity have been well documented in the grass carp, Ctenopharyngodon idella (Han et al 2010; Wu et al 2012). In addition, there are increasing amount of studies concerning to the gut microbiota in cyprinids using NGS approach as well in other fish species (Wu et al 2013; Ni et al 2014; Ye et al 2014; Kashinskaya et al 2015; Borsodi et al 2017; Li et al 2017a; Tran et al 2018a). However, there are only a few reports on Malaysian mahseer AACL Bioflux, 2021, Volume 14, Issue 1. 46 http://www.bioflux.com.ro/aacl (Tor tambroides) associated gut microbiome composition unlike other cyprinid. Our previous research by combination of traditional culture-depend method and PCR-DGGE revealed that gut bacterial community composition of cultured T. tambroides was continuously shifted in different developmental stages (Mohd Nosi et al 2018). Moreover, Tan et al (2019) showed different gut bacterial community composition between wild and captive T. tambroides and more diverse in wild T. tambroides gut bacterial community than captive one using NGS. Since T. tambroides is one of the most valuable freshwater fish with high demand in Malaysia and known as slow-growing species in the cyprinid family (Ng et al 2008), there are several researches that focused on their feed development especially dietary lipid composition (Ng et al 2011; Kamarudin et al 2018). To our concern, the information on gut microbiome of T. tambroides is still limited unlike other research like nutritional aspects and breeding. The aim of present study is to reveal comprehensive gut microbiome composition associated with cultured T. tambroides with different diets at different culture locations by 16S rRNA gene amplicon sequencing. Comparative analysis of different culture locations sample fed different diets might show core bacteria that could have high possibility to be utilized as probiotics to improve the growth of this slow growing carp. Material and Method Samples preparation. T. tambroides were collected from three different local farmer at Kota Bharu (6°4’30.245” N, 102°17’20.183” E) for Kelantan sample, Kuantan (3°47’39.811” N, 103°16’0.174” E) for Pahang sample, and Kuala Berang (4°53’15.173” N, 103°2’24.529” E) for Terengganu sample. Each T. tambroides was fed with combination of commercial pellet (Akafuji, JPD, Japan) and fruits (banana, papaya) for Kelantan sample, fed with combination of commercial pellet (AquaDine®, USA) and fruits (banana, grape) for Pahang sample, and fed with only vegetables such as cabbage and yardlong bean for Terengganu sample. Three healthy samples from each location; Kelantan (752.68±91.90 g), Pahang (1,158.34±201.27 g), and Terengganu (412.28±144.66 g) were randomly collected and anesthetized using a dose (100 mg L-1) of tricaine methanesulfonate (Finquel MS-222, Sigma-Aldrich, Missouri, USA). After removing any debris and external bacteria on fish body surface using sterile 0.01 M phosphate buffer saline (PBS; Sigma-Aldrich Cheme GmbH, Munich, Germany) and 70 % ethanol, whole gut was aseptically taken, washed three times with sterile 0.01 M PBS, transferred into sterile tubes, and stored at -80°C for 16S rRNA gene amplicon sequencing analysis. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. And the studies in this manuscript were approved by UMT Biosecurity and Ethics Committee (UMT/JKEPHMK/2020/45). DNA extraction, PCR amplification, Library preparation, and Bioinformatic analysis for 16S rRNA gene amplicon sequencing analysis using Illumina Miseq Platform. Promega DNA purification system (Promega, Madison, WI, USA) was used for bacterial genomic DNA from whole gut sample from each location. Then, DNA concentration and quality was evaluated by 1% w v-1 agarose gel electrophoresis, NanodropTM 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and Qubit® 2.0 Fluorometer (Invitrogen, Merelbeke, Belgium). Thereafter, each genomic DNA sample was amplified for V4 region of 16S rRNA gene with unique 8bp barcord containing universal primers (515F/806R), purified using Qiagen Gel Extraction kit (Qiagen, Germany), and then sequencing was conducted using the Illumina MiSeq platform (Illumina, Sandiego, USA) at Shanghai Megiddo Biological Pharmaceutical Co., Ltd. (Shanghai, China). Bioinformatics analyses were conducted using QIIME (Quantitative Insights Into Microbial Ecology) ver 1.9.1 (Caporaso et al 2010). Pairs of reads from the original DNA fragments were merged by PEAR. The fastq-formatted sequences were quality checked, and ambiguous sequences were filtered with quality score of 30 and 80 % coverage using FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and usearch (https://www.drive5.com/usearch/), respectively. After pre-processing, reference-based and chimera sequences were checked and removed from the cleaned sequences using AACL Bioflux, 2021, Volume 14, Issue 1. 47 http://www.bioflux.com.ro/aacl usearch61, and operational taxonomic unit (OTU) clustering was performed with a 0.97 threshold using uclust (Edgar 2010) and representative sequence were assigned to reference sequence in the SILVA database. Thereafter, five alpha diversity metrics (Observed OTUs, rarefaction curve, invsimpson, Chao1, and Shannon index) for each sample were estimated using QIIME. To evaluate the community divergence (beta diversity), we used Principal Coordinates Analysis (PCOA) based on weighted UniFrac distance to visualize the community composition differences among different culture locations using QIIME, and Venn diagrams to illustrate shared bacterial OTUs within same location sample and among different culture locations using MOTHUR (Schloss et al 2009). We also performed linear discriminant analysis effect size (LEfSe) (Segata et al 2011) to identify the differences of relative abundance of bacterial taxa among different culture location sample using the Huttenhower laboratory Galaxy web application (http://huttenhower.sph.harvard.edu/galaxy/). The Kruskal–Wallis

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